JP2004271540A - Protein chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein chip technique capable of fixing a protein on a substrate while keeping its activity without using an artificial synthesizing agent such as a supermolecular hydrogel-forming agent. <P>SOLUTION: (1) A glass substrate 1 is washed to remove impurities. (2) A sheet 2s of bacteria cellulose (BC) is stuck onto the surface of the glass substrate 1, whereby a GC layer 2 is formed on the glass substrate 1. (3) The BC layer 2 is worked into a BC 3 comprising array elements 3a arranged in a matrix by finely cutting it lengthwise and cross wise. (4) The generated dust is removed by laser beam emission. (5) The humidity of the BC 3 is adjusted. (6) An aqueous solution 6 containing the protein is dropped onto each array element 3a adjusted in humidity. (7) A protein chip in which a number of prob cells 3p consisting of the BC impregnated with the aqueous solution 6 containing the protein are arranged and fixed in a matrix on the glass substrate 1 is produced. As the BC, nata de coco is used. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a technique for identifying or analyzing a target molecule by observing an interaction between a probe biomolecule and a target molecule derived from a living body, and in particular, relates to a protein chip having at least a part of a protein as a probe and a protein chip. Related to the technology for manufacturing.

  With the completion of human genome sequence decoding and the spread of DNA chip technology (microarray method), genomic DNA expression analysis has been rapidly promoted, and a large amount of genomic information is being accumulated.

  However, in vivo, cell functions are regulated and maintained by proteins interacting with other proteins, small molecules, DNA, etc., and undergoing post-translational modifications such as phosphorylation. It is essential to analyze proteins involved in cell functions.

  Unlike DNA composed of four types of bases, proteins are composed of 20 types of amino acids and have a higher-order structure from a primary structure to a quaternary structure. Number.

  In addition, since there is no protein amplification method such as PCR for DNA and it is difficult to detect a control protein having a low expression level with high sensitivity, it is necessary to use a combination of several different analysis methods. Research on proteins requires labor and time, such as requiring skillful operations. Therefore, development of a high-throughput system for identifying a protein expressed in a living body in a short time or analyzing an interaction has been desired.

  Against this background, attempts have been made to apply the advantages of DNA chip technology, which enables comprehensive analysis, to proteome analysis. One of them is an analysis method using a protein chip (also called a protein array).

  There are roughly two types of protein chips. The first type is one in which a chemical treatment is applied to the substrate surface, that is, hydrophobic, ion-exchangeable, or metal ions are introduced into the substrate surface to form a number of spots having different properties, and a protein is applied to these spots. This is called a chemical chip, which is used for protein expression analysis, identification, and purification by utilizing the difference in physicochemical affinity of DNA. The second type is a biological chip in which biomolecules such as proteins, antibodies, and DNA are immobilized on the surface of a substrate, and the interacting proteins and ligands are searched for by utilizing the biochemical affinity of the proteins.

  In the analysis using a protein chip, after a protein is reacted with a large number of spots subjected to such chemical or biochemical modification, proteins and other contaminants showing no affinity are removed by washing, and TOF -MS (Time of Flight-mass Spectrometer, time-of-flight mass spectrometer) can measure the accurate molecular weight of the protein captured in the spot.

The protein chip can be applied to expression analysis and interaction analysis of proteins in a large number of kinds and a large amount of samples, as well as purification, identification, and functional analysis, and is expected as a platform technology for accelerating proteome analysis. In addition, research on sugar chain chips and cell chips for the purpose of analyzing the functions of sugar chains and cell functions has been promoted, and is attracting attention as a chip technology supporting post-genome research. (Non-Patent Document 1)
However, the DNA chip technology cannot be directly used for a protein chip. A DNA chip is one in which DNA or RNA is immobilized on a dried substrate, but most proteins function only in an aqueous solution, and the higher-order structure (three-dimensional structure) is lost at the moment of drying. Moreover, once the protein has been dried, it will not return to its original state even with water. Therefore, the protein cannot be dried and fixed on the substrate while maintaining its original higher-order structure. This is a big difference from DNA.

Against this background, a semi-wet device technology has been developed in which peptides and proteins are softly wrapped in a supramolecular hydrogel and immobilized on a substrate while maintaining activity. In this technique, an array of hydrogels is created by first heating and dissolving a supramolecular hydrogelator in a buffer solution, spotting it on a substrate such as glass, and cooling. Peptides and proteins are implanted into the semi-wet arrays of peptides and proteins. According to this technique, an array in which peptides and proteins are arranged can be easily created. (Non-Patent Document 2)
Supervised by Tomoji Kawai, "Dictionary of Nanotechnology", Industrial Research Committee, December 25, 2003, p853-854 Tadashi Hamachi, 2 others, Development of semi-wet peptide / protein using supramolecular hydrogel, "Bio Venture", Yodosha, April 20, 2004, Vol. 4 No. 3, p42-43

  The supramolecular hydrogelator used in the semi-wet device technology of Non-Patent Document 2 is a newly developed artificial synthetic agent.

  The problem to be solved by the present invention is to provide a protein chip technology capable of immobilizing a peptide or protein on a substrate while maintaining its activity without using an artificial synthetic agent such as a supramolecular hydrogelator. It is in.

  In order to solve the above problems, the present invention employs the following solutions.

  The method for producing a protein chip according to the present invention includes a step of forming a bacterial cellulose layer on a substrate, a step of processing the bacterial cellulose layer into a bacterial cellulose array comprising arrayed array elements, and a method of preparing a protein (probe biomolecule). Impregnating (dropping, spotting) the array element with an aqueous solution containing

  Another method for producing a protein chip according to the present invention includes a step of processing a sheet of bacterial cellulose into a bacterial cellulose array comprising arrayed array elements; a step of fixing the bacterial cellulose array on a substrate; Impregnating the array element with an aqueous solution containing a biomolecule).

  Still another method for producing a protein chip according to the present invention includes a step of forming a bacterial cellulose layer on a substrate and a step of orderly arranging and spotting an aqueous solution containing a protein (probe biomolecule) on the bacterial cellulose layer. Including.

  The protein chip according to the present invention is obtained by immobilizing probe cells made of bacterial cellulose impregnated with an aqueous solution containing a protein (probe biomolecule) on a substrate in an orderly manner.

  In another protein chip according to the present invention, a bacterial cellulose layer is formed on a substrate, and an aqueous solution containing a protein (probe biomolecule) is neatly arranged and spotted on the bacterial cellulose layer.

  The plate for preparing a protein chip according to the present invention is obtained by fixing bacterial cellulose before containing the aqueous solution on a substrate.

  The protein retaining method according to the present invention is characterized in that the protein is retained in a state of being suspended in an aqueous solution existing between fibrils of bacterial cellulose.

  Here, “orderly arranged” means that the elements are regularly arranged in a fixed arrangement such as a line, a matrix, and a honeycomb. Since the array elements and the probe cells of the bacterial cellulose layer are regularly arranged in a predetermined alignment state, addressing of the probe cells (array elements) and automatic spotting of the probe biomolecule aqueous solution become possible.

  Nata de coco is most suitable as the bacterial cellulose used in the present invention. Nata de coco is a harmless material that can be eaten. Nata de coco has high shape retention performance (strong stiffness). Nata de coco is easy to process such as slicing thinly and cutting finely. Nata de coco can be stored for a long time if kept in a sterile condition. Nata de coco can be easily cultivated at room temperature without requiring special production equipment, so that the production cost is low. Nata de coco is natural cellulose and therefore friendly to the natural environment.

  Bacterial cellulose is cellulose produced by natural cellulose-producing bacteria. Examples of cellulose-producing bacteria include acetic acid bacteria such as Acetobactor acetosum, Acetobactor xylinum, Acetobactor pasterianum, and Acetobactor lancens, and Sarcina ventriculi. Examples include Bacterium xyloides, Pseudomonas, Agrobacterium, and the like. Acetobactor aceti var. Xylinum is most preferred as a bacterial cellulose producing bacterium used in the present invention. Acetobactor aceti var. Xylinum is a bacterium that has long been used in the Philippines and other countries as a fibrous thick film producing bacterium called nata.

  Bacterial cellulose can hold several times to several hundred times the weight of water with respect to the cellulose component. Bacterial cellulose has, as a skeleton, a fibril net that is three-dimensionally stretched. Bacterial cellulose containing water retains most of its water within the network of fibril nets. When an aqueous solution containing a protein is impregnated with bacterial cellulose, most of the aqueous solution is retained in the mesh of the fibril net. Since the protein in the aqueous solution is suspended in the aqueous solution that fills the mesh of the fibril net, the protein retains its original higher-order structure.

  Therefore, according to the production method of the present invention, a bacterial cellulose layer is formed on a substrate, the bacterial cellulose layer is processed into a bacterial cellulose array comprising arrayed array elements, and an aqueous solution containing a protein is dropped on each array element. By doing so, proteins can be arranged and fixed on the substrate in order while maintaining their activity.

  Further, according to another production method of the present invention, a bacterial cellulose layer is formed on a substrate, and an aqueous solution containing the protein is neatly arranged and spotted on the bacterial cellulose layer to spot the protein on the substrate while maintaining its activity. It can be arranged and fixed in order.

  There are several ways to form a bacterial cellulose layer on a substrate. One example is a method in which bacterial cellulose (nata) taken out of a culture tank is sliced into a sheet and attached to a substrate. At this time, it is desirable that proteins, DNAs, and the like derived from the producing bacteria existing in the bacterial cellulose be removed using an appropriate treating agent. The bacterial cellulose sheet may be dried and then attached to a substrate such as glass, or may be attached to the substrate in a wet state. The bacterial cellulose sheet may be dried on the attached substrate.

  When drying bacterial cellulose, it is desirable to prevent adhesion of fibrils in the cellulose. As a method therefor, there is a method of freeze-drying or solvent-substitutingly drying an aqueous suspension of bacterial cellulose so as not to cause a bond derived from a hydrogen bond between fibrils at the time of drying. Further, a method of adding a third component other than water to an aqueous suspension containing bacterial cellulose followed by dehydration drying (Japanese Patent Laid-Open No. 09-165402), or a method of dehydrating and drying bacterial cellulose produced by stirring culture under tension. And a method of disintegrating the obtained bacterial cellulose (WO97 / 48730).

  As another method of forming a bacterial cellulose layer on a substrate, there is a method of culturing bacterial cellulose directly on a substrate. If a biopolymer such as a cellulose material is used for the substrate, and a rail made of a substance having an affinity for the cellulose-producing bacterium is formed on the surface of the substrate, the cellulose-producing bacterium runs along the rail and the fibrils , A cellulose film having high orientation can be formed. Alternatively, a large number of pits (small depressions) may be formed on the surface of the substrate in an orderly manner, and bacterial cellulose may be directly cultured in each pit.

  There are several ways to process a bacterial cellulose layer into a bacterial cellulose array consisting of ordered array elements. For example, there is a method of cutting / dividing a sheet-shaped bacterial cellulose attached to a substrate into array elements using a laser beam or the like. By cutting the bacterial cellulose adhered to the substrate with a laser beam, fine and precise processing can be performed. Alternatively, the sheet-like bacterial cellulose may be divided into array elements at a place different from the substrate and then attached to the substrate.

  According to the production method of the present invention, a protein chip in which a protein is immobilized on a substrate while maintaining its activity can be produced without using an artificial synthetic agent such as a supramolecular hydrogelator.

  According to the protein chip of the present invention, since the protein is retained on the substrate while maintaining its activity, the interaction analysis and screening between the protein on the substrate and a molecule derived from a living body can be performed accurately and efficiently. Can be implemented.

  According to the plate for producing a protein chip of the present invention, the protein chip of the present invention can be easily produced.

  According to the protein retention method of the present invention, the protein is retained in a state of being suspended in an aqueous solution existing between fibrils of bacterial cellulose, so that the protein retains its activity without destroying its higher-order structure. Can be held on the substrate.

The best mode for carrying out the present invention will be described with reference to the drawings. In a plurality of drawings, common constituent elements or substantially the same constituent elements are denoted by the same reference numerals, and description thereof will be omitted as appropriate.
[Producing method of protein chip]
1.1 Manufacturing method 1
1.1.1 Configuration FIG. 1 is a manufacturing process diagram showing an example of a method for manufacturing a protein chip according to the present invention.

  (1) Substrate cleaning step: In this step, the glass substrate 1 is cleaned to remove impurities.

  (2) Bacterial cellulose layer forming step: In this step, a bacterial cellulose layer 2 composed of bacterial cellulose sheet 2s is formed on the glass substrate 1 by attaching a bacterial cellulose (BC) sheet 2s to the surface of the glass substrate 1. Form.

  The sheet 2s is obtained by slicing bacterial cellulose (nata) taken out of the culture tank into a sheet. The dead bodies of the production bacteria, proteins, DNAs, etc., which were present in the bacterial cellulose during the culturing, have been removed by placing the bacterial cellulose in a clean water stream. When it is not possible to remove dead bacteria and the like by the water flow alone, a method of immersing bacterial cellulose in a solution of an appropriate treating agent and washing with water is employed. Examples of the treating agent include proteolytic enzymes, DNA degrading enzymes, dilute acids, alkalis, sodium hypochlorite, hydrogen peroxide, and surfactants (sodium lauryl sulfate, deoxycholic acid, etc.). Further, heating and cleaning in a range from room temperature to 200 ° C. is also effective.

  (3) Bacterial cellulose array forming step: In this step, the bacterial cellulose layer 2 is processed into a bacterial cellulose array 3 composed of array elements 3a arranged in a matrix by finely cutting the layer with a laser beam 4 vertically and horizontally. By using an automatic fine processing device (not shown) that can accurately control the irradiation point of the laser beam 4, the bacterial cellulose layer 2 can be precisely processed into the bacterial cellulose array 3.

  (4) Cleaning step: In this step, dust generated by laser beam irradiation (particles such as slag and ash due to laser beam irradiation) is removed by any method such as washing with water, blowing air, or applying vibration. This step may be performed in parallel with the step of forming a bacterial cellulose array. Removal of dead bacteria and the like of the production bacteria may be performed in this step.

  (5) Humidity adjustment step: In this step, the humidity (humidity, water content) of the bacterial cellulose array 3 is adjusted. Here, the modes of humidity control include water removal, that is, drying and water addition. Drying means not only a state of being dry to almost no moisture, but also a state of fresh drying (a state in which some water remains) or a state in which the moisture content is less than immediately after being taken out of water. This is a broad concept that includes If the water content is about 25% or less based on the weight of the solid content (mainly fibrils) contained in the dried product, it can be said that the product is almost dry.

  When the bacterial cellulose array 3 is dried, measures are taken to prevent adhesion of fibrils in cellulose constituting each array element 3a. As a method therefor, there is a method of freeze-drying or solvent-substitutingly drying an aqueous suspension of bacterial cellulose so as not to cause a bond derived from a hydrogen bond between fibrils at the time of drying. Further, a method of adding a third component other than water to an aqueous suspension containing bacterial cellulose followed by dehydration drying (Japanese Patent Laid-Open No. 09-165402), or a method of dehydrating and drying bacterial cellulose produced by stirring culture under tension. And a method of disintegrating the obtained bacterial cellulose (WO97 / 48730).

  (6) Probe biomolecule fixing step: In this step, an aqueous solution 6 containing a protein (receptor, hormone, enzyme, antibody, etc.) is dropped on each array element 3a of the bacterial cellulose array 3 whose humidity has been adjusted. At this time, a plurality of types of aqueous solutions 6 containing different proteins are prepared in advance, and the aqueous solutions 6 containing different proteins are dropped on each array element 3a. The aqueous solution 6 dropped on each array element 3a permeates the bacterial cellulose constituting the array element 3a. As a result, the proteins in the dropped aqueous solutions 6 are fixed on the glass substrate 1 while being held in the respective array elements 3a.

  In this step, each aqueous solution 6 can be accurately dropped on each array element 3 a of the bacterial cellulose array 3 by using an automatic dropping device capable of accurately controlling the dropping point and the drop amount of each aqueous solution 6. The amount of protein retained in each array element 3a can be freely adjusted by controlling the protein concentration in each aqueous solution 6 and the amount of the aqueous solution dropped onto each array element 3a. As an automatic dropping device (spotter), a known arrayer (Japanese Patent Application Laid-Open No. 10-503841, Japanese Patent Application No. 2002-507386, Japanese Patent Application No. 2003-515113, Japanese Patent Application No. 2004-512514), which is used when manufacturing a spot-type DNA chip, 2003-156492, JP-A-2003-344014, JP-A-2004-045241, etc.) can be used. In order not to damage the bacterial cellulose array 3, it is desirable to use a non-contact type spotter. When using a pin type spotter, it is necessary to optimize the Spot-Pin-Over Travel value.

(7) Post-treatment step: Through the above series of steps, a large number of probe cells 3p made of bacterial cellulose (array element 3a) impregnated with the aqueous solution 6 containing the protein are arranged and fixed on the glass substrate 1 in a matrix. A protein chip 7 is manufactured (see FIG. 5). The manufactured protein chip 7 is housed in a synthetic resin package (casing) as required.
1.1.2 Action / Effect Bacterial cellulose can hold water several times to several hundred times the weight of cellulose components. Bacterial cellulose has, as a skeleton, a fibril 2f net (fibril net) spread three-dimensionally in a complicated manner (see FIG. 2A). Bacterial cellulose containing water retains most of its water within the network of fibril nets. When bacterial solution is impregnated with the aqueous solution 6 containing the protein 5, most of the aqueous solution 6 is retained in the mesh of the fibril net (FIG. 2 (b)). Since the protein 5 in the aqueous solution 6 is suspended in the aqueous solution 6 filling the mesh of the fibril net, the original three-dimensional structure of the biomolecule can be maintained.

  Therefore, a bacterial cellulose layer 2 is formed on a glass substrate 1, and the bacterial cellulose layer 2 is processed into a bacterial cellulose array 3 composed of array elements 3a arranged in a matrix. By dropping the protein 5 on the glass substrate 1, the protein 5 can be arranged and fixed on the glass substrate 1 in a matrix while maintaining its activity.

  By using the protein chip 7 manufactured in this manner, a protein or other molecule that binds to the protein 5 on the glass substrate 1 can be efficiently screened.

In the above-described example, the protein chip including the bacterial cellulose array 3 including the array elements 3a arranged in a matrix has been described. However, the array elements 3a are arranged so as to be in another alignment state such as a line shape or a honeycomb shape. It is also possible (see FIG. 6).
1.2 Manufacturing method 2
1.2.1 Configuration FIG. 3 is a manufacturing process diagram showing another example of a method for manufacturing a protein chip according to the present invention.

  (1) Substrate cleaning step: In this step, the glass substrate 1 is cleaned to remove impurities.

  (2) Bacterial cellulose array forming step: In this step, a bacterial cellulose (BC) sheet 2s (see FIG. 1) is finely cut vertically and horizontally using a laser beam 4 or the like to form an array arranged in a matrix. It is processed into a bacterial cellulose array 3 composed of elements 3a.

  (3) Bacterial cellulose array fixing step: In this step, the bacterial cellulose array 3 is fixed on the surface of the washed glass substrate 1.

  (4) Humidity adjustment step: In this step, the humidity (humidity, water content) of the bacterial cellulose array 3 is adjusted.

  (5) Probe biomolecule fixing step: In this step, an aqueous solution 6 containing a protein (receptor, hormone, enzyme, antibody, etc.) is dropped on each array element 3a of the bacterial cellulose array 3 whose humidity has been adjusted. At this time, a plurality of types of aqueous solutions 6 containing different proteins are prepared in advance, and the aqueous solutions 6 containing different proteins are dropped on each array element 3a. The aqueous solution 6 dropped on each array element 3a permeates the bacterial cellulose constituting the array element 3a. As a result, the proteins in the dropped aqueous solutions 6 are fixed on the glass substrate 1 while being held in the respective array elements 3a.

(6) Post-treatment step: Through the above series of steps, a large number of probe cells 3p made of bacterial cellulose (array element 3a) impregnated with the aqueous solution 6 containing the protein are arranged and fixed on the glass substrate 1 in a matrix. A protein chip 7 is manufactured (see FIG. 5). The manufactured protein chip 7 is housed in a synthetic resin package (casing) as required.
1.2.2 Function / Effect Bacterial cellulose can hold water several times to several hundred times the weight of cellulose components. Bacterial cellulose has, as a skeleton, a fibril 2f net (fibril net) spread three-dimensionally in a complicated manner (see FIG. 2A). Bacterial cellulose containing water retains most of its water within the network of fibril nets. When bacterial solution is impregnated with the aqueous solution 6 containing the protein 5, most of the aqueous solution 6 is retained in the mesh of the fibril net (FIG. 2 (b)). Since the protein 5 in the aqueous solution 6 is suspended in the aqueous solution 6 filling the mesh of the fibril net, the original three-dimensional structure of the biomolecule can be maintained.

  Therefore, a sheet of bacterial cellulose is processed into a bacterial cellulose array 3 composed of array elements 3a arranged in a matrix, fixed on the glass substrate 1, and an aqueous solution 6 containing protein 5 is dropped on each array element 3a. The protein 5 can be arranged and fixed on the glass substrate 1 in a matrix while maintaining its activity.

  By using the protein chip 7 manufactured in this manner, a protein or other molecule that binds to the protein 5 on the glass substrate 1 can be efficiently screened.

In the above-described example, the protein chip including the bacterial cellulose array 3 including the array elements 3a arranged in a matrix has been described. However, the array elements 3a are arranged so as to be in another alignment state such as a line shape or a honeycomb shape. It is also possible (see FIG. 6).
1.3 Manufacturing Method 3
1.3.1 Configuration FIG. 4 is a manufacturing process diagram showing still another example of the method for manufacturing a protein chip according to the present invention.

  (1) Substrate cleaning step: In this step, the glass substrate 1 is cleaned to remove impurities.

  (2) Bacterial cellulose layer forming step: In this step, the bacterial cellulose layer 2 composed of the bacterial cellulose sheet 2s is formed on the glass substrate 1 by attaching a bacterial cellulose (BC) sheet 2s to the surface of the glass substrate 1. Form.

  (3) Humidity adjustment step: In this step, the humidity (humidity, water content) of the bacterial cellulose layer 2 is adjusted.

  (4) Probe biomolecule fixing step: In this step, an aqueous solution 6 containing a protein is spotted in a matrix on the bacterial cellulose layer 2 whose humidity has been adjusted.

(5) Post-processing step: a protein chip 8 having a large number of detection spots (probe cells) 2sp formed by spotting an aqueous solution 6 containing a protein in a matrix on the bacterial cellulose layer 2 by the above series of steps. (See FIG. 7). The manufactured protein chip 8 is housed in a synthetic resin package (casing) or the like as necessary.
1.3.2 Action / Effect Bacterial cellulose can hold water several times to several hundred times the weight of cellulose components. Bacterial cellulose has, as a skeleton, a fibril 2f net (fibril net) spread three-dimensionally in a complicated manner (see FIG. 2A). Bacterial cellulose containing water retains most of its water within the network of fibril nets. When bacterial solution is impregnated with the aqueous solution 6 containing the protein 5, most of the aqueous solution 6 is retained in the mesh of the fibril net (FIG. 2 (b)). Since the protein 5 in the aqueous solution 6 is suspended in the aqueous solution 6 filling the mesh of the fibril net, the original three-dimensional structure of the biomolecule can be maintained.

  Therefore, the bacterial cellulose layer 2 is formed on the glass substrate 1 and the aqueous solution 6 containing the protein 5 is spotted on the bacterial cellulose layer 2 in the form of a matrix. It can be arranged and fixed in a shape.

  By using the protein chip 8 manufactured as described above, it is possible to efficiently screen for proteins and other molecules that bind to the protein 5 on the glass substrate 1.

In the above example, a protein chip in which the detection spots 2sp are arranged in a matrix has been described. However, the detection spots 2sp can be arranged in another alignment state such as a line shape or a honeycomb shape (FIG. 8).
[Protein chip]
2.1 Protein chip 1
2.1.1 Configuration FIG. 5 is a plan view showing an embodiment of a protein chip according to the present invention. The protein chip 7 is formed by arranging a large number of probe cells 3p made of bacterial cellulose impregnated with an aqueous solution 6 containing a protein in a matrix on the glass substrate 1. The protein chip 7 is manufactured through a series of steps shown in FIG.
2.1.2 Action / Effect In each probe cell 3p of the protein chip 7, the protein 5 is held while maintaining its activity in a state of being suspended in the aqueous solution 6 (FIGS. 2A and 2B). )reference).

  Therefore, by using this protein chip 7, it is possible to efficiently screen for proteins and other molecules that bind to the protein 5 on the glass substrate 1.

In the above-described example, the protein chip 7 including the bacterial cellulose array 3 including the probe cells 3p arranged in a matrix has been described. However, as shown in FIG. 6, the protein chip 7 has another alignment state such as a line shape or a honeycomb shape. The protein chip in which the probe cells 3p (3a) are arranged as described above is also included in the protein chip of the present invention.
2.2 Protein chip # 2
2.2.1 Configuration FIG. 7 is a plan view showing an embodiment of the protein chip according to the present invention. The protein chip 8 has a large number of detection spots 2sp formed by spotting an aqueous solution 6 containing a protein on the bacterial cellulose layer 2 in a matrix. The protein chip 8 is manufactured through a series of steps shown in FIG.
2.1.2 Action / Effect In each detection spot 2sp of the protein chip 8, the protein 5 is retained while maintaining its activity in a state of being suspended in the aqueous solution 6 (FIGS. 2A and 2B). )reference).

  Therefore, by using this protein chip 8, it is possible to efficiently screen for proteins and other molecules that bind to the protein 5 on the glass substrate 1.

In the above example, the protein chip 8 having the detection spots 2sp arranged in a matrix has been described. However, as shown in FIG. 8, the detection spots 2sp are arranged so as to be in another alignment state such as a line shape or a honeycomb shape. Such a protein chip is also included in the protein chip of the present invention.
2.3 Protein chip # 3
2.3.1 Configuration FIG. 9A is a plan view showing an embodiment of a protein chip according to the present invention, and FIG. 9B is a longitudinal sectional view. The protein chip 9 has a large number of probe cells 3p made of bacterial cellulose (BC) impregnated with an aqueous solution 6 containing a protein, arranged and fixed on the glass substrate 1 in a matrix. On the surface of the glass substrate 1 of the protein chip 9, a large number of hemispherical pits 1p are formed in a matrix. The protein chip 9 is manufactured by directly culturing bacterial cellulose (BC) in each pit 1p, and then impregnating the bacterial cellulose (BC) in each pit 1p with an aqueous solution 6 containing a protein. That is, the aqueous solution-containing bacterial cellulose (BC) in each pit 1p constitutes the probe cell 3p.
2.3.2 Action / Effect In each probe cell 3p of the protein chip 9, the protein 5 is held while maintaining its activity in a state of being suspended in the aqueous solution 6 (FIGS. 2A and 2B). )reference).

  Therefore, by using the protein chip 9, it is possible to efficiently screen for proteins and other molecules that bind to the protein 5 on the glass substrate 1.

In the above example, the probe cells 3p are formed in the pits 1p arranged in a matrix. However, the probe cells 3p are formed in the pits 1p arranged in another alignment state such as a line shape or a honeycomb shape. The formed protein chip is also included in the protein chip of the present invention.
[Plate for making protein chips]
3.1 Substrate part 1
3.1.1 Configuration FIG. 10 is a plan view showing an embodiment of a plate for producing a protein chip according to the present invention. The protein chip making plate 11 is formed by forming a bacterial cellulose array 3 on a glass substrate 1 and comprising a large number of array elements 3a arranged in a matrix. The protein chip making plate 11 is manufactured by passing through a substrate washing step, a bacterial cellulose layer forming step, a bacterial cellulose array forming step, a cleaning step, and a humidity adjusting step in FIG. The protein chip making plate 11 is housed in a synthetic resin package (casing) as required.
3.1.2 Function / Effect According to the plate 11 for preparing protein chips, the aqueous solution 6 containing a protein is dropped on each array element 3a of the bacterial cellulose array 3 fixed on the glass substrate 1 ( The protein chip 7 shown in FIG. 5 can be easily prepared (corresponding to the probe biomolecule fixing step in FIG. 1).

  In the above example, the plate 11 for producing protein chips provided with the bacterial cellulose array 3 composed of the array elements 3a arranged in a matrix has been described. However, the array elements may be arranged in another alignment state such as a line shape or a honeycomb shape. The plate for preparing a protein chip on which 3a is arranged is also included in the plate for preparing a protein chip of the present invention.

Further, a product manufactured by omitting the humidity adjustment step may be used as the plate 11 for preparing a protein chip. In that case, it is desirable to adjust the humidity of the bacterial cellulose array 3 when dropping the aqueous solution 6 containing the protein.
3.2 Board 2
3.2.1 Configuration FIG. 11 is a plan view showing another embodiment of the plate for producing a protein chip according to the present invention. The plate 12 for producing a protein chip is formed by forming a bacterial cellulose layer 2 on a glass substrate 1. The plate 12 for producing a protein chip is manufactured through a substrate washing step, a bacterial cellulose layer forming step, and a humidity adjusting step in FIG. The protein chip making plate 12 is housed in a synthetic resin package (casing) as necessary.
3.2.2 Function / Effect According to the plate 12 for preparing protein chips, the aqueous solution 6 containing the protein 5 is spotted in a matrix on the bacterial cellulose layer 2 (probe biomolecule immobilization in FIG. 4). 7), and the protein chip 7 of FIG. 7 can be easily produced.

  Further, by spotting the aqueous solution 6 containing the protein 5 on the bacterial cellulose layer 2 so as to be in another alignment state such as a line shape or a honeycomb shape, the other solution such as a line shape or a honeycomb shape as shown in FIG. A protein chip in which the detection spots 2s are arranged so as to be aligned can be easily created.

Further, a plate manufactured for omitting the humidity control step may be used as the protein chip forming plate 12. In this case, it is desirable to adjust the humidity of the bacterial cellulose array layer 2 when the aqueous solution 6 containing the protein is dropped.
3.3 Board 3
3.3.1 Configuration FIG. 12A is a plan view showing still another example of the plate for producing a protein chip according to the present invention, and FIG. 12B is a longitudinal sectional view. The protein chip making plate 13 is formed by forming a large number of pits 1p in a matrix on the surface of the glass substrate 1 and directly culturing bacterial cellulose (BC) in each pit 1p. The plate 13 for producing a protein chip is manufactured through a substrate washing step, a bacterial cellulose layer forming step, and a humidity control step. The protein chip making plate 13 is housed in a synthetic resin package (casing) as necessary.
3.3.2 Function / Effect According to the plate 13 for preparing protein chips, the protein chip 9 of FIG. 9 is easily prepared by impregnating the bacterial cellulose (BC) of each pit 1p with the aqueous solution 6 containing the protein. can do.

The plate manufactured for omitting the humidity control step may be used as the protein chip forming plate 13. In this case, it is desirable to adjust the humidity of bacterial cellulose (BC) in the pit 1p when the aqueous solution 6 containing the protein is dropped.
[Supplemental explanation]
In the above description, a probe cell holding a protein as a probe biomolecule or a protein chip in which only a detection spot is formed on a glass substrate has been described. However, a probe cell containing a protein and other substances (objects) such as peptides and DNA A protein chip of the present invention also includes a hybrid type protein chip in which probe cells holding RNA, metal ions, cells, and the like are formed on the same substrate (see FIG. 13).

  Further, in the above example, a glass substrate was used as the substrate of the protein chip and the chip-making plate, but a synthetic resin substrate, a metal substrate, a bacterial cellulose substrate, or a substrate of another material may be used. .

  The planar shape of each probe cell (array element 3a) of the bacterial cellulose array does not need to be square. The shape may be rectangular, triangular, pentagonal, hexagonal, octagonal, circular, or elliptical, but is preferably circular (see FIG. 14).

  The cell size and cell interval of the probe cells (array elements) are arbitrary. The cell size (diameter) is preferably selected from the range of tens of microns to several millimeters. It is desirable that the cell size and the cell interval can be spotted by a ready-made arrayer.

  The thickness of the bacterial cellulose layer or the probe cell (array element) is arbitrary. Preferably, it is selected from a range of ten and several microns to ten and several millimeters in a wet state.

  Further, the shape (planar shape) of the protein chip and the chip making plate is arbitrary. Any shape such as a square, a rectangle, a circle, an ellipse, and a trapezoid may be used.

  The protein chip of the present invention can be used as a cell array by introducing or culturing living cells into bacterial cellulose on the substrate. That is, the cDNA clone inserted into the expression vector is placed on bacterial cellulose (for example, the array element 3a of the chip making plate 11 shown in FIG. 10, the bacterial cellulose layer 2 of the chip making plate 12 shown in FIG. 11, etc.). By spotting, growing cells thereon, and introducing the arrayed genes, a cell array in which a foreign gene is expressed can be prepared. Techniques such as a fluorescent immunostaining method and a FISH method can be used for cells on the cell array. Bacterial cellulose is extremely high in water content, and can retain water and nutrients required for cell culture for a long time.

  The protein chip of the present invention can be used as a tissue array by placing a biological tissue specimen (tissue) on bacterial cellulose on the substrate. Bacterial cellulose has an extremely high water content, so that a biological tissue specimen can be kept in a suitable wet state for a long time.

  The protein chip of the present invention can be used as a sugar chain array by impregnating bacterial cellulose on the substrate with an aqueous solution containing a sugar chain.

Manufacturing process diagram showing an example of a method for manufacturing a protein chip according to the present invention (A) Schematic diagram showing the internal structure of bacterial cellulose (b) Schematic diagram showing how proteins are held in bacterial cellulose in a state of being suspended in an aqueous solution Manufacturing process diagram showing an example of a method for manufacturing a protein chip according to the present invention Manufacturing process diagram showing an example of a method for manufacturing a protein chip according to the present invention FIG. 2 is a plan view showing an embodiment of a protein chip according to the present invention. (A) Plan view showing a form example of a protein chip in which probe cells are arranged in a line shape. (B) Plan view showing a form example of a protein chip in which probe cells are arranged in a honeycomb form. FIG. 2 is a plan view showing an embodiment of a protein chip according to the present invention. (A) Plan view showing a form example of a protein chip in which detection spots are arranged in a line shape. (B) Plan view showing a form example of a protein chip in which detection spots are arranged in a honeycomb form. (A) A plan view showing an embodiment of a protein chip according to the present invention. (B) A longitudinal sectional view showing an embodiment of a protein chip according to the present invention. FIG. 2 is a plan view showing an embodiment of a plate for producing a protein chip according to the present invention. FIG. 2 is a plan view showing an embodiment of a plate for producing a protein chip according to the present invention. (A) A plan view showing an embodiment of a plate for producing a protein chip according to the present invention. (B) A longitudinal sectional view showing an embodiment of a plate for producing a protein chip according to the present invention. A conceptual diagram showing an example of a form of a protein chip according to the present invention. FIG. 2 is a plan view showing an embodiment of a protein chip or a chip-making plate according to the present invention.

Explanation of reference numerals

Reference Signs List 1 glass substrate 1p pit 2 bacterial cellulose layer 2f fibril 2s sheet 2sp detection spot 3 bacterial cellulose array 3a array element 3p probe cell 4 laser beam 5 protein 6 aqueous solution 7 protein chip 8 protein chip 9 protein chip 11 plate for preparing protein chip 12 protein Chip making plate 13 Protein chip making plate BC Bacterial cellulose

Claims (10)

Forming a bacterial cellulose layer on the substrate,
Processing the bacterial cellulose layer into a bacterial cellulose array consisting of ordered array elements,
Impregnating the array element with an aqueous solution containing a protein,
A method for producing a protein chip, comprising: Processing the bacterial cellulose sheet into a bacterial cellulose array consisting of ordered array elements;
Fixing the bacterial cellulose array on a substrate,
Impregnating the array element with an aqueous solution containing a protein,
A method for producing a protein chip, comprising: Forming a bacterial cellulose layer on the substrate,
Spotting an aqueous solution containing a protein in an orderly arrangement on the bacterial cellulose layer,
A method for producing a protein chip, comprising:   The method according to any one of claims 1 to 3, wherein the bacterial cellulose is nata de coco.   A protein chip in which probe cells made of bacterial cellulose impregnated with an aqueous solution containing a protein are arranged and fixed on a substrate in an orderly manner.   A protein chip in which a bacterial cellulose layer is formed on a substrate, and an aqueous solution containing a protein is arranged and spotted on the bacterial cellulose layer in an orderly manner.   7. The protein chip according to claim 5, wherein the bacterial cellulose is nata de coco. A plate for producing a protein chip according to any one of claims 5 to 7,
A plate for preparing a protein chip in which bacterial cellulose before being impregnated with the aqueous solution is fixed on a substrate.   A protein retention method, wherein a protein is retained in a suspended state in an aqueous solution existing between fibrils of bacterial cellulose. The protein retention method according to claim 9, wherein the bacterial cellulose is nata de coco.

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